PROJECT SUMMARY Gastro-intestinal (GI) cancer is a significant public health burden in the US. Among GI cancers, colorectal cancer (CRC) has been a focus of attention because it is the third most common cancer in the US. CRC arises through a polyp to cancer progression sequence, driven by molecular alterations (gene mutations and epigenetic alterations) in the normal colon cells, as well as by other factors including the tumor microenvironment, gut microbiome, etc. The identification of these molecular alterations and determination of their role in the etiology and behavior of CRC is incomplete and under active investigation. Another GI cancer that has received considerable attention recently is esophageal adenocarcinoma (EAC) because of its three-fold increase in the last few decades and because of its poor prognosis (20% 5-year survival). Like CRC, EAC arises secondary to gene mutations and epigenetic alterations that drive the formation of a pre-malignant condition called Barretts esophagus (BE) that can then progress to EAC. Although progress has been made in the prevention and management of GI cancers, there remains a substantial need to advance our understanding of the molecular pathology of GI cancers and to develop biomarkers that will translate into improved cancer screening programs, better patient management and outcomes. In order to address this need, the Research Specialist Dr. Ming Yu has developed this research plan to achieve the following objectives: 1) Characterize and functionally interrogate epigenetic alterations, in particular DNA methylation changes, during the initiation and progression of GI cancer; 2) Assess methylated genes as potential molecular biomarkers. This research plan is an integral part of the research programs that the Unit Director, Dr. William Grady, has developed at the Fred Hutchinson Cancer Research Center (FHCRC) (PQC1R01CA194663; PQ6R01CA220004), and several multi-disciplinary NCI funded research networks (the Early Detection Research Network (EDRN; U01 CA086389- 08), the Barrett?s Esophagus Translational Research Network (BETRNet; 5U54CA163060), and a Bridging the Gap UO1 (5U01CA182940)). Furthermore, this plan represents unique lines of investigation that Dr. Yu has initiated and will be leading in the next five years. These new investigations include the characterization of enhancer methylomes, the DNA methylation changes at gene enhancer elements, during GI cancer imitation and progression; the identification and the functional validation of onco-enhancers, and development of organoids derived from normal and cancerous gastrointestinal tissues to study DNA methylation during cancer initiation and progression. These approaches will provide an exciting addition to the existing research programs. Dr. Yu, the Research Specialist in this proposal, is the principal scientist successfully leading the studies of these research programs in the Grady laboratory at the FHCRC and has been the central determinant of success of the Grady lab over the last few years. Dr. Yu has a central role as an ?integrating node? scientist in the EDRN, BETRNET and other multi-investigator team projects. In this Research Plan, Dr. Yu will conduct a comprehensive assessment of DNA methylation changes during cancer initiation and progression, using whole genome bisulfite sequencing (WGBS), which provides unprecedented coverage of CpGs across genomes at single CpG resolution. She will integrate WGBS data with RNA-seq, CHIP-seq and clinical data of the same samples to gain insights into the features of a candidate onco-enhancer, and test the innovative hypothesis that aberrant DNA methylation at enhancer regions in cancer is the predominant way aberrant methylation in cancer regulates gene expression. To elucidate whether DNA methylation at candidate oncoenhancers affects target gene expression, she will develop CRISPR-Cas9 based DNA methylation editing tools to target enhancer regions and assess the biological relevance of DNA methylation alterations in cells from normal and cancerous tissues. Of note, she has established methods to culture 3-dimentional organoids from primary colon epithelium and CRC, which represent the most relevant ex vivo culture system to date. Moreover, she will also discover and validate promising methylated gene biomarkers that will function as biomarkers for BE and EAC, as well as CRC field cancerization loci. Dr. Yu will capitalize on her productive collaborations in the EDRN and BTRNET to successfully develop these biomarker assays. Prominently, she will apply a novel technology MethyLight Droplet Digital PCR that she developed to advance the identification of an innovative set of methylated DNA markers of colon cancer risk, termed ?field cancerization markers?. Successful completion of the proposed studies will lead to novel insight into the roles of epigenetic alterations in molecular pathogenesis of GI cancer. Furthermore, these findings will be translated into the discovery of DNA methylation biomarkers that can improve the management of GI cancer.